Compositions and methods for treatment of lymphatic and venous vessel arterialization

Abstract

The present invention is directed to methods and compositions that may be used in disrupting the association of smooth muscle cells with lymphatic endothelial cells and in correcting the valvular dysfunction in veins and lymphatic vessels. Such compositions are useful for therapeutic and prophylactic treatment of impaired lymphatic and venous function, particularly for the treatment of lymphedema distichiasis or chronic venous insufficiency.

1 . A therapeutic or prophylactic method of improving lymphatic function comprising: 
 administering to a mammalian subject a composition comprising an inhibitor of arterialization of lymphatic vessels,    wherein the subject is selected from: subjects with impaired lymphatic function due to arterialization of lymphatic vessels, and subjects with a genetic risk for developing said impaired function.    
     
     
         2 . The method of  claim 1 , wherein the subject is human.  
     
     
         3 . The method of  claim 2 , wherein the subject has lymphedema from the impaired lymphatic function.  
     
     
         4 . The method of  claim 2 , wherein the subject has lymphedema distichiasis.  
     
     
         5 . The method of  claim 2 , wherein the impaired lymphatic function is diagnosed from abnormal lymphatic drainage, increased number of lymph nodes, lymph flowback, and a lymphatic capillary network that comprises smooth muscle cells associated with lymphatic cells.  
     
     
         6 . The method of  claim 2 , wherein the subject has a Foxc2 mutation genotype.  
     
     
         7 . The method of  claim 2 , wherein the subject has lymphatic vessels characterized by at least one arterialization indicator selected from the group consisting of: 
 (A) expression of a smooth muscle cell marker in lymphatic capillaries from the subject;    (B) tortuous and distended dermal lymphatic capillaries;    (C) smooth muscle cells associated with lymphatic vessels;    (D) pericytes associated with lymphatic vessels;    (E) lymphatic hyperplasia; and,    (F) expression in lymphatic vessels of a blood vessel basal lamina proteins.    
     
     
         8 . The method of  claim 2 , wherein the subject has lymphatic vessels characterized by absent or dysfunctional lymphatic valves.  
     
     
         9 . The method of  claim 2 , wherein the subject has a genetic risk for developing lymphedema, wherein said risk is diagnosed 
 from determinations that the subject has one or more of the following: (A) at least one genetic relative with hereditary lymphedema; (B) a Foxc2 mutation genotype; (C) a Foxc1 mutation genotype; (D) distichiasis; (E) smooth muscle cells covering lymphatic capillaries; (F) hyperplastic cutaneous lymphatic vessel density; (G) PDGFR-beta-expressing cells associated with lymphatic vessels; (H) PDGF-B overexpression in lymphatic vessels (I) smooth muscle cell association with lymphatic capillaries; (J) pericyte association with lymphatic capillaries; and (K) expression of blood vessel basal lamina components in lymphatic vessels.    
     
     
         10 . The method of  claim 9 , wherein said risk is further diagnosed from an absence of or dysfunctional lymphatic valves in lymphatic vessels of the subject.  
     
     
         11 . The method of  claim 9 , wherein the subject has at least one genetic relative with hereditary lymphedema and has a Foxc2 mutation genotype.  
     
     
         12 . The therapeutic method according to  claim 2 , wherein the inhibitor of arterialization is administered in an amount effective to reduce edema, reduce pain from edema, increase limb function, increase FOXC2 expression in lymphatic endothelia, reduce PDGF-B expression in lymphatic endothelia, or reduce SMC association with lymphatic capillaries, or combinations thereof.  
     
     
         13 . The therapeutic method according to  claim 2 , wherein the inhibitor of arterialization is administered in an amount effective to reduce lymphatic backflow as judged by lymphoscintigraphy.  
     
     
         14 . The prophylactic method according to  claim 2 , wherein the inhibitor of arterialization is administered in an amount effective to inhibit smooth muscle cell or pericyte association with lymphatic capillaries in the subject.  
     
     
         15 - 16 . (canceled)  
     
     
         17 . The method according to  claim 1 , wherein the inhibitor of arterialization is an inhibitor of PDGFR-beta activity.  
     
     
         18 . The method of  claim 17 , wherein the inhibitor of PDGFR-beta activity is selected from the group consisting of: 
 (A) inhibitors of PDGFR-beta expression;    (B) inhibitors of PDGF-B stimulation of PDGFR-beta;    (C) inhibitors of PDGF-B expression;    (D) inhibitors of PDGFR-beta signaling; and    (E) combinations thereof.    
     
     
         19 . The method of  claim 18 , wherein the inhibitor of PDGFR-beta activity is an inhibitor of PDGFR-beta expression selected from the group consisting of: 
 (A) an antisense molecule directed to PDGFR-beta;    (B) an interfering RNA (RNAi) directed to PDGFR-beta;    (C) an aptamer that binds PDGFR-beta RNA;    (D) a ribozyme directed to PDGFR-beta; and    (E) combinations thereof.    
     
     
         20 . The method of  claim 18 , wherein the inhibitor of PDGFR-beta activity is an inhibitor of PDGF-B stimulation of PDGFR-beta selected from the group consisting of: 
 (A) an antibody substance that binds to the extracellular domain of PDGFR-beta and inhibits PDGF binding;    (B) an antibody substance that binds to PDGF-B and inhibits the PDGF-B from binding or activating PDGFR-beta;    (C) a polypeptide comprising a soluble fragment of PDGFR-beta, wherein the polypeptide and fragment bind PDGF-B;    (D) a fragment of PDGF-B that binds and fails to stimulate PDGFR-beta;    (E) a polypeptide comprising a soluble fragment of PDGFR-alpha, wherein the polypeptide and fragment bind PDGF-B; and    (F) combinations thereof.    
     
     
         21 . The method of  claim 18 , wherein the inhibitor of PDGFR-beta activity is an inhibitor of PDGF-B expression selected from the group consisting of: 
 (A) an antisense molecule directed to PDGF-B;    (B) interfering RNA (RNAi) directed to PDGF-B;    (C) an aptamer that binds PDGF-B RNA;    (D) a ribozyme directed to PDGF-B; and    (E) combinations thereof.    
     
     
         22 . (canceled)  
     
     
         23 . The method of  claim 18 , wherein the inhibitor of PDGFR-beta activity is a tyrosine kinase inhibitor.  
     
     
         24 . The method of  claim 23 , wherein the tyrosine kinase inhibitor is imatinib mesylate.  
     
     
         25 . The method of  claim 2 , wherein the inhibitor of arterialization is administered subcutaneously at a site of edema.  
     
     
         26 . A method according to  claim 2 , further comprising administering to the subject a growth factor product selected from the group consisting of vascular endothelial growth factor C (VEGF-C) protein products, vascular endothelial growth factor D (VEGF-D) protein products, VEGF-C gene therapy products, and VEGF-D gene therapy protein products.  
     
     
         27 . A method according to  claim 2 , further comprising administering to the subject a smooth muscle cell relaxant.  
     
     
         28 . A therapeutic or prophylactic method of treating arterialization of lymphatic vessels in a mammalian subject, comprising: 
 providing isolated lymphatic endothelial cells or lymphatic endothelial progenitor cells;    transforming or transfecting the cells ex vivo with a polynucleotide comprising a nucleotide sequence that encodes an inhibitor of PDGF expression; and    administering the transformed or transfected cells to the mammalian subject.    
     
     
         29 . A method according to  claim 28 , wherein the transformed or transfected cells are administered locally at a site of edema in the subject.  
     
     
         30 . A therapeutic or prophylactic method of improving lymphatic function comprising: 
 administering to a mammalian subject a composition comprising a smooth muscle relaxant;    wherein the subject is identified as having arterialization of lymphatic vessels; and    wherein the smooth muscle relaxant is administered in an amount effective to improve lymphatic function in the subject.    
     
     
         31 . The method of  claim 30 , wherein the composition is administered locally at a site of edema in the subject.  
     
     
         32 . A method of screening for an agent to improve lymphatic function in mammalian subjects having arterialization of lymphatic vessels, comprising steps of: 
 contacting arterialized lymphatic tissue with a test agent, wherein the arterialized lymphatic tissue comprises lymphatic endothelial cells associated with pericytes or smooth muscle cells;    determining if the test agent causes dissociation of lymphatic endothelial cells from pericytes or smooth muscle cells, wherein a test agent that causes the dissociation is selected as an agent to improve lymphatic function.    
     
     
         33 . The method of  claim 32 , wherein the lymphatic tissue is obtained from an organism with a Foxc2 mutation.  
     
     
         34 . The method of  claim 32 , further comprising a step of contacting blood vessels with the test agent, and selecting a test agent that preferentially causes smooth muscle dissociation form lymphatic vessels compared to dissociation of smooth muscle cells from blood vessels.  
     
     
         35 . A therapeutic or prophylactic method of improving lymphatic function comprising: 
 isolating lymphatic endothelial cells or lymphatic endothelial progenitor cells from a subject selected from: subjects with impaired lymphatic function due to arterialization of lymphatic vessels, and subjects with a genetic risk for developing said impaired function;    transforming or transfecting the cells ex vivo with a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence at least 90% identical to the Foxc2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide is expressed in the cells and has Foxc2 transcription factor activity; and    administering the transformed or transfected cells to the mammalian subject, in an amount effective to reduce or prevent lymphatic backflow in a lymphatic vessel, thereby improving lymphatic function.    
     
     
         36 - 37 . (canceled)  
     
     
         38 . A method according to  claim 35 , further comprising a step of coating a synthetic valve with the transformed or transfected cells, wherein the administering comprises implanting the synthetic valve into a vessel in the mammalian subject.  
     
     
         39 . A therapeutic or prophylactic method of improving lymphatic function comprising: 
 administering a composition comprising a polynucleotide to a subject, wherein the subject is selected from: subjects with impaired lymphatic function due to arterialization of lymphatic vessels, and subjects with a genetic risk for developing said impaired function; and wherein the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence at least 90% identical to the Foxc2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide has Foxc2 transcription factor activity.    
     
     
         40 . The method of  claim 39 , wherein said composition is administered locally at a site in need of treatment to improve lymph flow.  
     
     
         41 . The method of  claim 39 , wherein the composition comprises an expression vector that comprises an expression control sequence operatively linked to the polynucleotide.  
     
     
         42 . (canceled)  
     
     
         43 . The method of  claim 39 , wherein the polynucleotide further comprises a promoter that promotes expression of the polynucleotide in a mammalian cell.  
     
     
         44 . An improvement to a synthetic valve for implantation in a lumen of a blood or lymphatic vessel, said improvement comprising coating a surface of the valve with endothelial cells, wherein the endothelial cells are transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that comprises an amino acid sequence at least 90% identical to the Foxc2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide is expressed in the cells and has Foxc2 transcription factor activity.  
     
     
         45 . An isolated endothelial cell or endothelial precursor cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that comprises an amino acid sequence at least 90% identical to the Foxc2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide is expressed in the cell and has Foxc2 transcription factor activity.  
     
     
         46 . A medical device comprising: a synthetic valve that is implantable in a mammalian vessel; and endothelial cells according to  claim 45  on a surface of the synthetic valve.  
     
     
         47 . A medical device comprising an endovascular stent that is implantable in a mammalian vessel, and endothelial cells according to  claim 43  on a surface of the stent.  
     
     
         48 . A therapeutic or prophylactic method of improving venous flow comprising: 
 isolating venous endothelial cells or venous endothelial progenitor cells from a mammalian subject selected from: subjects with impaired venous flow due to absent or dysfunctional venous valves, and subjects with a genetic risk for developing said impaired flow;    transforming or transfecting the cells ex vivo with a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence at least 90% identical to the FOXC2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide is expressed in the cells and has FOXC2 transcription factor activity; and    administering the transformed or transfected cells to the mammalian subject, in an amount effective to reduce or prevent venous backflow in a blood vessel, thereby improving venous function.    
     
     
         49 - 51 . (canceled)  
     
     
         52 . A method according to  claim 48 , wherein the subject has a FoxC2 mutation genotype.  
     
     
         53 . (canceled)  
     
     
         54 . A method according to  claim 48 , wherein a suspension of the cells is administered intravenously into the mammalian subject.  
     
     
         55 . A method according to  claim 48 , further comprising a step of coating a synthetic valve with the transformed or transfected cells, wherein the administering comprises implanting the synthetic valve into a vein in the mammalian subject.  
     
     
         56 . A therapeutic or prophylactic method of improving venous flow comprising: 
 administering a composition comprising a polynucleotide to a subject, wherein the subject is selected from: subjects with impaired venous flow due to the absence of or dysfunctional venous valves, and subjects with a genetic risk for developing said impaired function; and wherein the polynucleotide comprises a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence at least 90% identical to the FOXC2 amino acid sequence of SEQ ID NO: 2 or a fragment thereof, wherein the polypeptide has Foxc2 transcription factor activity.    
     
     
         57 . The method of  claim 56 , wherein said composition is administered locally at a site in need of treatment to improve venous flow.  
     
     
         58 . The method of  claim 56 , wherein the composition comprises an expression vector that comprises an expression control sequence operatively linked to the polynucleotide.  
     
     
         59 . (canceled)  
     
     
         60 . The method of  claim 56 , wherein the polynucleotide further comprises a promoter that promotes expression of the polynucleotide in a mammalian cell.